Proteomic Profiling of Cardiomyocyte-Specific Cathepsin A Overexpression Links Cathepsin A to the Oxidative Stress Response

Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to β-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography–tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated <i>p</i>-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response

Proteomic Profiling of Cardiomyocyte-Specific Cathepsin A Overexpression Links Cathepsin A to the Oxidative Stress Response

Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to β-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography–tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated <i>p</i>-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response

Cathepsin H function in production of lung surfactant proteins.

<p>(<b>A</b>) mRNA expression of surfactant proteins A1, B, and C measured by quantitative ‘real-time’ RT-PCR in lungs of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice (n = 5 per group). (<b>B</b>) Detection of surfactant protein B (SP-B) in lung tissue lysates (<b>C</b>) Western blot detection of SP-B in broncho-alveolar lavage (BAL) of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice of 2 genetic backgrounds (129P2/OlaHsd and C57BL/6N). The lysosomal membrane associated protein 2a (Lamp 2a) is present at the limiting membrane of lamellar bodies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026247#pone.0026247-Wasano1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026247#pone.0026247-Albrecht1" target="_blank">[28]</a> and serves as loading control independent of the surfactant proteins. (<b>D</b>–<b>F</b>) Surface activity of BAL fluid measured by pulsating bubble surfactometry (n = 6–10).</p

Targeted disruption of the cathepsin H (Ctsh) gene.

<p>(<b>A</b>) Scheme for the targeted disruption of mouse Ctsh gene by homologous recombination. Exons are indicated by number. (<b>B</b>) Southern blot analysis of SacI-digested genomic DNA from mouse liver by the 5′ external probe denoted in panel A. Expected fragment sizes are 5.5 kb for wild-type and 6.8 kb for mutant <i>Ctsh</i> alleles. (<b>C</b>) Northern blots from liver and kidney samples of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice. The Ctsh 5′ probe detects the genuine 1.6 kb mouse Ctsh transcript in the <i>Ctsh^+/+</i> samples. *Denotes an enlarged transcript in <i>Ctsh</i>^−/− consisting of Ctsh exons 1–5 plus lacZ reporter.</p

Cathepsin expression and gross phenotype of Ctsh-deficient mice.

<p>(<b>A</b>) Western blots for Ctsh detection in lungs and liver of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice. (<b>B</b>) Detection of “acidic” aminopeptidase activity at pH 6.0 in lungs, livers and kidneys of f <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice (n = 3). (<b>C</b>) Observed and expected genotype frequencies of litters from <i>Ctsh</i>^+/− x <i>Ctsh</i>^+/− matings. (<b>D</b>) Weight gain of female littermates from heterozygous matings (n = 5 per genotype). (<b>E</b>) mRNA expression of cathepsin C (Ctsc) and cathepsin E (Ctse) measured by quantitative ‘real-time’ RT-PCR in lungs of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice (n = 5 per group). (<b>F</b>) Cathepsin C (Ctsc) and cathepsin E (Ctse) detected by Western blotting in <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− lung lysates.</p